TECHNICAL FIELD
[0001] The present invention relates to an antibody-drug conjugate comprising a drug conjugated
to the N-terminal amino acid residue of the heavy chain or light chain of an antibody,
a preparation method thereof, and the use thereof.
BACKGROUND ART
[0002] In recent years, methods of diagnosing or treating various diseases using antibodies
have been studied. Particularly, because of the target specificity of antibodies,
various therapeutic methods using antibodies have been developed, and various types
of drugs containing antibodies, for example, antibody-drug conjugates (ADCs), have
been developed. Thus, studies have been continuously conducted to increase the
in vivo stability of antibodies or antibody-drug conjugates and maximize the therapeutic
effects thereof.
[0003] Among them, antibody-drug conjugates generally have the disadvantage of low
in vivo stability compared to natural antibodies, but have been developed in order to overcome
the disadvantages (low therapeutic effects) of natural antibodies by conjugating them
to drugs. Various antibody-drug conjugates in which drugs having certain medical effects,
such as cytotoxin, are conjugated to target-specific antibodies, have been developed.
In particular, a method of inducing cancer cell death by cytotoxin conjugated to a
cancer cell-specific antibody is a method that is actually currently used.
[0004] However, such antibody-drug conjugates generally have low
in vivo stability compared to natural antibodies. Furthermore, if drug antibody ratio (DAR)
is increased in order to increase therapeutic effects, there will be various technical
problems to be solved. First, an increase in drug antibody ratio should not interfere
with the antigen-binding ability and Fc function of antibodies for target-specific
therapy, should lead to an increase in therapeutic effects, and should not reduce
the
in vivo stability (i.e., blood half-life) of antibody-drug conjugates. The object of the
current antibody-drug conjugate preparation field is to maintain the highest possible
antibody drug ratio in view of the above-described technical problems. In particular,
considering that the expression level of cancer cell surface antigens is low, the
highest possible drug antibody ratio (DAR) should be maintained in order to maintain
high cytotoxicity. However, if DAR reaches 8, there is a problem in that the blood
half-life of the antibody-drug conjugate decreases due to the effect of the hydrophobic
drug conjugated to the antibody so that the toxicity thereof can increase and
in vivo efficacy thereof can decrease.
[0005] Under this background, the present inventors have made extensive efforts to develop
a technology capable of preparing an antibody-drug conjugate which maintains the antigen-binding
activity of a parent antibody, exhibits excellent anticancer effects, and has low
drug toxicity and excellent
in vivo efficacy. As a result, the present inventors have found that, when a drug is conjugated
to the N-terminus of the heavy chain or light chain of an antibody, the antibody-drug
conjugate has excellent blood stability and anticancer activity while having low
in vivo toxicity compared to previously reported antibody-drug conjugates, thereby completing
the present invention.
DISCLOSURE OF INVENTION
[0006] It is an object of the present invention to provide an antibody-drug conjugate comprising
a drug conjugated to the N-terminal amino acid residue of the heavy chain or light
chain of an antibody.
[0007] Another object of the present invention is to provide a method for preparing the
above antibody-drug conjugate.
[0008] Still another object of the present invention is to provide a composition comprising
the above antibody-drug conjugate.
[0009] Yet another object of the present invention is to provide a method for treating cancer,
comprising administering the above antibody-drug conjugate to a subject suspected
of having cancer.
[0010] A further object of the present invention is to provide a method for treating autoimmune
disease, comprising administering the above antibody-drug conjugate to a subject suspected
of having autoimmune disease.
[0011] A still further object of the present invention is to provide a method for screening
an antibody suitable for use in preparation of the above antibody-drug conjugate.
ADVANTAGEOUS EFFECTS
[0012] A method for preparing an antibody-drug conjugate according to the present invention
can prepare an antibody-drug conjugate having higher
in vivo efficacy, stability and lower toxicity.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013]
FIG. 1 shows the structural formula of toxin monomethyl auristatin F (MMAF) having
an aldehyde linker connected to the end.
FIG. 2 is a schematic diagram showing the structure of a non-genetically modified
monoclonal antibody-cytotoxin conjugate in which the number and site of cytotoxin
moieties conjugated to an antibody are homogeneous.
FIG. 3 shows the LC/MS profile of T-N-MMAF.
FIG. 4 shows the results of peptide mapping performed to determine the site of binding
of a drug in a prepared Trastuzumab-N-MMAF (T-N-MMAF conjugate).
FIG. 5 shows the results of SEC-HPLC analysis of a prepared T-N-MMAF.
FIG. 6 shows a time-dependent change in the blood concentration of total antibody
in rats.
FIG. 7 shows a time-dependent change in the blood concentration of conjugated antibody.
FIG. 8 shows a comparison of the PK profiles of total antibody and conjugated antibody
between antibody-drug conjugates (ADCs).
FIG. 9 shows growth curves of tumors formed by the HCC1954 cell line in nude rat xenograft
models.
FIG. 10 shows survival curves obtained in nude rat xenograft model experiments performed
to measure the tumor volume at the endpoint.
FIG. 11 shows the change and relative change in weight by administration of each antibody-drug
conjugate (ADC).
FIG. 12 shows the results of examining whether the administration of each ADC caused
hepatotoxicity.
FIG. 13 shows the changes in neutrophils and platelets by administration of each ADC.
FIG. 14 shows the results of LC/MS analysis of T-N-MMAE.
FIG. 15 shows the rat PK profile of T-N-MMAE.
FIG. 16 shows the LC/MS profile of Brentuximab-N-MMAF (B-N-MMAF).
FIG. 17 shows the results of analyzing the antigen-binding activity of B-N-MMAF.
FIG. 18 shows the conjugation profile of Lorvotuzumab-N-MMAF (L-N-MMAF).
FIG. 19 shows the antigen-binding activity of L-N-MMAF.
BEST MODE FOR CARRYING OUT THE INVENTION
[0014] In one aspect, the present invention provides is directed to an antibody-drug conjugate
comprising a drug conjugated to the N-terminal amino acid residue of the heavy chain
or light chain of an antibody.
[0015] As used herein, the term "antibody-drug conjugate (ADC)" refers to the form in which
a drug and an antibody are chemically conjugated to each other without reducing the
biological activities of the antibody and the drugs. In the present invention, the
term "antibody-drug conjugate" refers to the form in which the drug is conjugated
to the N-terminal amino acid residue of the heavy chain and/or light chain of the
antibody, particularly, the form in which the drug is conjugated to the N-terminal
α-amine group of the heavy chain and/or light chain of the antibody. In the present
invention, it was found that, when a drug was site-specifically conjugated to the
N-terminus of the heavy chain or light chain among various regions of an antibody,
the antibody-drug conjugate had excellent
in vivo efficacy and stability and low toxicity, compared to previously reported antibody-drug
conjugates, including antibody-drug conjugates formed by cysteine conjugation, antibody-drug
conjugates formed by thiol conjugation, and antibody-drug conjugates formed by lysine
conjugation, indicating that the N-terminus of the heavy chain or light chain of antibodies
can be a site advantageous in terms of efficacy, stability and low toxicity. This
schematic view of an antibody-drug conjugate according to the present invention is
schematically shown in FIG. 2.
[0016] As used herein, the term "N-terminus" refers to the amino terminus (N-terminus) of
the heavy chain or light chain of an antibody, which is a site to which a drug can
be conjugated for the purpose of the present invention. Examples of the N-terminus
include, but are not limited to, not only amino acid residues at the distal end of
the N-terminus, but also amino acid residues near the N-terminus. Specifically, the
term "N-terminus" refers to the first amino acid residue of the heavy chain or light
chain of an antibody, and more specifically, refers to the α-amine group of the first
amino acid of the heavy chain or light chain of an antibody, but is not limited thereto.
[0017] The antibody-drug conjugate according to the present invention can have the advantage
of guaranteeing homogeneity through the site-specific conjugation or number-specific
conjugation between an antibody and a drug. Particularly, through the optimization
procedure of the present invention, 1-8 drug moieties corresponding to the optimal
drug-antibody ratio (DAR) can be conjugated to the N-terminal amino acid residue of
each antibody molecule.
[0018] As used herein, the term "homogeneity" refers to the case in which the ratio and
site of conjugation between two substances in a conjugate of the two substances are
homogeneous. However, the term is intended to include not only the case in which the
ratio and site of conjugation are completely homogeneous, but also the case in which
a specific ratio and site of conjugation are predominant. When a conjugate has homogeneity,
it is entirely homogeneous, and the dose-dependent efficacy thereof can be accurately
measured, and thus the dose and number of administration thereof can be standardized.
[0019] As used herein, the term "antibody" means a protein molecule which comprises an immunoglobulin
molecule immunologically reactive with a certain antigen, and which serves as a receptor
that specifically recognizes the antigen. The term is intended to encompass polyclonal
antibodies, monoclonal antibodies, full-length antibodies and antibody fragments containing
antigen binding domains. A full-length antibody has two full-length light chains and
two full-length heavy chains, in which each of the light chains is linked to the heavy
chain by a disulfide bond. The full-length antibody comprises IgA, IgD, IgE, IgM and
IgG, and subtypes of IgG include IgG1, IgG2, IgG3 and IgG4. The term "antibody fragment"
refers to a fragment having an antigen-binding function, and is intended to include
Fab, Fab', F(ab')
2, scFv and Fv. Fab comprises light-chain and heavy-chain variable regions, a light-chain
constant region, and a heavy-chain first constant domain (CH1), and has one antigen-binding
site. Fab' differs from Fab in that it has a hinge region including one or more cysteine
residues at the C-terminus of the heavy-chain CH1 domain. An F(ab')
2 antibody is formed by a disulfide bond between the cysteine residues of the hinge
region of Fab'. Fv means a minimal antibody fragment having only a heavy-chain variable
region and a light-chain variable region. dsFv is has a structure in which a heavy-chain
variable region and a light-chain variable region are linked to each other by a disulfide
bond, and scFV generally has a structure in which a heavy-chain variable region and
a light-chain variable region are covalently linked to each other by a peptide linker.
These antibody fragments can be obtained using proteases (for example, Fab fragments
can be obtained by digesting a full-length antibody with papain, and F(ab')
2 fragments can be obtained by digesting a full-length antibody with pepsin). Preferably,
these antibody fragments can be produced by a genetic recombinant technique. These
antibody fragments can be obtained using proteases (for example, digestion of a whole
antibody with papain or pepsin affords Fab or F(ab')2, respectively), and preferably
may be constructed by genetic recombination techniques.
[0020] In addition, the antibody that is used in the present invention may be a natural
antibody or a recombinant antibody. As used herein, the term "natural antibody" refers
to an antibody that has undergone no genetic modification. The natural antibody may
have a significantly low risk of immunogenicity, unlike antibodies genetically modified
in vivo. As used herein, the term "recombinant antibody" means a genetically modified antibody
which may have an antigen-binding activity or desired characteristic imparted by genetic
modification.
[0021] As used herein, the term "genetic modification" refers to an action of changing the
amino acid sequence of interest and is intended to include the modification of polypeptides
having amino acid sequences that somewhat differ from the amino acid sequence of a
native sequence polypeptide encoding the amino acid sequence of interest. Amino acid
sequence variants contain an amino acid sequence having a substitution, deletion or
insertion of one or more amino acid residues at one or more specific positions in
a native amino acid sequence.
[0022] The antibody that is used in the present invention may be an antibody recognizing
a cell surface antigen which is internalized into cells when binding to the antibody.
For the purpose of the present invention, when an antigen is internalized into cells
by binding to the antibody, a drug, particularly a cytotoxic drug, conjugated to the
antibody, can enter the cells due to the characteristics of the antibody, and thus
can exhibit high efficacy, but is not limited thereto.
[0023] In addition, the antibody that is used in the present invention may be an antibody
that binds specifically to a cancer cell surface antigen or a surface antigen of a
tissue in which an autoimmune disease has occurred.
[0024] As used herein, the term "cancer cell surface antigen" refers to either a substance
that is not produced in normal cells or not exposed to the cell surface, or a substance
that is exposed to the cell surface specifically in cancer cells, or s substance that
is present more on the surface of cancer cells than on the surface of normal cells.
When the substance of interest is recognized by the antibody, it is referred to as
an antigen.
[0025] Specifically, the cancer cell surface antigen that is used I the present invention
may be any cancer cell surface antigen that can be recognized specifically by the
antibody of the present invention. Examples of the cancer cell surface antigen may
include CD19, CD20, CD30, CD33, CD37, CD22, CD56, CD70, CD74, CD138, Muc-16, mesothelin,
HER2, HER3, GPNMB(glycoprotein NMB), IGF-1R, BCMA(B cell maturation antigen), PSMA(prostate-specific
membrane antigen), EpCAM(Epithelial cell adhesion molecule), and EGFR (epidermal growth
factor receptor). More specifically, the cancer cell surface antigen may be any one
selected from the group consisting of HER2, CD30, CD56, and GPNMB, but is not limited
thereto. In an example of the present invention, Trastuzumab, a kind of anti-HER2
antibody, Lorvotuzumab, a kind of anti-CD56 antibody, Brentuximab, a kind of anti-CD30
antibody, and Glembatumumab, a kind of anti-GPNMB antibody, which recognize Her2,
CD56 and GPNMB, were used as model antibodies.
[0026] As used herein, the term "drug" means any substance having cell-specific biological
activity, and is intended to include compounds, DNA, RNA, peptides and the like. The
term "drug" is intended to include not only substances containing a reactive group
capable of crosslinking with an α-amine group, but also substances having a linker
containing a reactive group capable of crosslinking with an α-amine group. In this
case, the drug can bind site-specifically to the N-terminal amino acid residue of
the antibody by the linker, but is not limited thereto.
[0027] The term "linker" refers to a chemical moiety comprising an atomic chain that allows
the drug to bind covalently to the antibody. The linker is prepared in a state in
which it is connected to the drug, and the end of the linker has a reactive group
that can be linked to the antibody.
[0028] Examples of the reactive group capable of crosslinking with an α-amine group include
any reactive groups known in the art, which can crosslink with the N-terminal α-amine
group of the heavy chain or light chain of the antibody. Examples of the reactive
group may include isothiocyanate, isocyanate, acyl azide, NHS ester, sulfonyl chloride,
aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, imidoester, carbodiimide,
anhydride, and fluorophenyl ester. More preferably, the reactive group is aldehyde
or NHS ester, but not specifically limited thereto. Such reactive groups can be bound
with the amine group by acylation or alkylation, but are not specifically limited
thereto.
[0029] In particular, the antibody-drug conjugate of the present invention may be an immunoconjugate
in which the drug connected with a linker having a reactive aldehyde group is conjugated
to the N-terminal amino acid residue of the antibody in a site-specific and number-specific
way.
[0030] The reactive aldehyde group is effective in site-specifically conjugating the drug
to the N-terminal amino acid residue (particularly α-amine) of the antibody while
minimizing non-specific reactions. The final product produced through reductive alkylation
by an aldehyde bond is much more stable than that linked by an amide bond. The reactive
aldehyde group has the property of selectively reacting with the N-terminal α-amine
at low pH. Thus, the conjugate of the present invention has homogeneous in that the
drug is site-specifically conjugated to the N-terminal α-amine of the antibody. Thus,
the present invention overcomes the problem of the prior art in which the uniform
efficacy and quality of a drug cannot be guaranteed because of heterogeneity of the
number and site of conjugations in conventional antibody-drug conjugates, but is not
specifically limited thereto.
[0031] In an example of the present invention, the present inventors have found that, when
a conjugation reaction is carried out at a pH of 6.0 or lower in order to conjugate
a cytotoxic drug site-specifically to the α-amine of an antibody, the conjugation
of the cytotoxic drug to the ε-amine of lysine residues can be minimized.
[0032] The drug that is used in the present invention may be any substance that can induce
the activation or inhibition of certain signaling pathways, including cell death,
cell proliferation, immune activation and immune suppression. In particular, the drug
may be a cytotoxic drug or an immunosuppressive agent.
[0033] As used herein, the term "cytotoxic drug" refers to any substance, for example, a
compound, which has a cytotoxic or cell proliferation inhibitory effect. The term
"cytotoxic effect" refers to the effect of inhibiting or reducing the function of
cells to induce disruption of the cells, and the term "cell proliferation inhibitory
effect" refers to the effect of limiting cell growth functions such as cell growth
or cell proliferation.
[0034] Examples of the cytotoxic drug that is used in the present invention include chemotherapeutic
agents, including microtubule structure formation inhibitors, meiosis inhibitors,
RNA polymerase inhibitors, topoisomerase inhibitors, DNA intercalators, DNA alkylators
and ribosome inhibitors, protein toxins that can function as enzymes, and radioisotopes.
Examples of the cytotoxic drug may include maytansinoid, auristatin, dolastatin, tubulysin,
calicheamicin, pyrrolobenzodiazepines, doxorubicin, duocamycin, carboplatin(paraplatin),
cisplatin, cyclophosphamide, ifosfamide, nidran, [nitrogen mustard(mechlorethamine
HCL)], bleomycin, mitomycin C, cytarabine, fluorouracil, gemcitabine, trimetrexate,
methotrexate, etoposide, vinblastine, vinorelbine, alimta, altretamine, procarbazine,
taxol, taxotere, topotecan, irinotecan, trichothecene, CC1065, alpha-amanitin, other
enediyne antibiotics, exotoxin, and plant toxin. In addition, compounds include their
stereoisomers and derivatives. Furthermore, the auristatin that is used in the present
invention may be monomethyl auristatin E or monomethyl auristatin F, but is not limited
thereto.
[0035] The term "immunosuppressive agent" refers to any compound having the effect of reducing
immune responses. The term means a substance that can antagonize immune causing substances
or that can inhibit substances (cytokines such as interleukins) which are involved
in immune responses.
[0036] In an example of the present invention, Trastuzumab, Lorvotuzumab, Brentuximab and
Glembatumumab were used as model antibodies, and monomethyl auristatin E ((MMAE))
or monomethyl auristatin F (MMAF) were used as cytotoxic drugs to be conjugated to
the N-terminus of the antibodies (Examples 1 and 2). Trastuzumab was allowed to react
with MMAF or MMAE at a pH of 6.0 to conjugate the drug to the N-terminus of the antibody,
thereby preparing an antibody-drug conjugate. In the case of this antibody-drug conjugate,
it was shown that the antigen binding activity of the antibody and the cytotoxic efficacy
of the drug were maintained even after conjugation of the drug (Examples 3 to 5).
In particular, this antibody-drug conjugate showed excellent stability in human serum
in vitro compared to another antibody-drug conjugate (comparative conjugate) having a cysteine
or lysine bond, and also showed excellent pharmacokinetics in an excellent pharmacokinetic
experiment performed using rats (Example 6). In addition, this antibody-drug conjugate
showed excellent anticancer activity compared to the comparative conjugate in anticancer
animal models, but showed low toxicity similar to a control group in terms of weight,
hepatotoxicity, blood and the like (Examples 7 and 8). Furthermore, results similar
to the above-described results in terms of antigen binding activity, cytotoxicity
and the like were obtained even when other drugs such as MMAE were used or when other
antibodies such as Lorvotuzumab, Brentuximab and Glembatumumab were used (Example
9), indicating that the technology according to the present invention, in which a
drug is conjugated to the N-terminus of the heavy chain or light chain of an antibody,
can become a platform technology in the preparation of antibody-drug conjugates.
[0037] In another aspect, the present invention is directed to a method for preparing the
antibody-drug conjugate.
[0038] The antibody-drug conjugate and its components are as described above.
[0039] Specifically, the method for preparing the antibody-drug conjugate comprises allowing
an antibody to react with a drug containing a reactive group capable of crosslinking
with an α-amine group, thereby conjugating the drug to the N-terminal α-amine group
of the heavy chain or light chain of the antibody.
[0040] In addition, the method for preparing the antibody-drug conjugate may further comprise
separating the antibody-drug conjugate from a reaction product including the antibody
and the drug, which did not form the conjugate.
[0041] Specifically, in the preparation method, the antibody and the drug may be conjugated
to each other at a pH of 4.0-6.5, more specifically 5.5-6.5, even more specifically
6.0. As described above, the present invention has an advantage in that specific conjugation
between an aldehyde group present in the drug or its linker and the N-terminal α-amine
of the antibody can occur at low pH.
[0042] The process of separating the antibody-drug conjugate can be performed by various
methods known in that art. For example, it can be performed by a chromatographic process
including size exclusion chromatography, but is not specifically limited thereto.
[0043] In still another aspect, the present invention is directed to a composition comprising
the antibody-drug conjugate.
[0044] The composition may be in the form of a pharmaceutical composition for treating cancer
or autoimmune disease, which comprise the antibody-drug conjugate. In this case, the
antibody may be an antibody that binds specifically to a cancer cell surface antigen
or a surface antigen of a tissue in which autoimmune disease has occurred. The pharmaceutical
composition of the present invention may further comprise a pharmaceutically acceptable
carrier.
[0045] The antibody, the drug, the cancer cell surface antigen and the surface antigen of
the tissue in which autoimmune disease has occurred are as described above.
[0046] As used herein, the term
"cancer" includes all the kinds of cancers without limitations, but examples of the cancer
may include esophageal cancer, stomach cancer, large intestine cancer, rectal cancer,
oral cancer, pharynx cancer, larynx cancer, lung cancer, colon cancer, breast cancer,
uterine cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, testis
cancer, bladder cancer, renal cancer, liver cancer, pancreatic cancer, bone cancer,
connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's
disease, lymphoma, and multiple myeloid blood cancer. A cancer that can be treated
depending on the kind of an antigen specific to the cancer cell surface antigen may
be selected.
[0047] The term "autoimmune disease", as used herein, refers to any autoimmune disease that
is targeted by the antibody-drug conjugate. Examples of the autoimmune disease include
rheumatoid arthritis, systemic scleroderma, systemic lupus erythematosus, atomic dermatitis,
psoriasis, alopecia areata, asthma, Crohn's disease, Behcet's disease, Sjögren's syndrome,
Guillain-Barre syndrome, chronic thyroiditis, multiple sclerosis, polymyositis, ankylsoing
spondylitis, fibrositis, and polyarteritis nodosa.
[0048] As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier
or diluent that does not impair the biological activity and characteristics of an
administered compound without irritating an organism. As a pharmaceutically acceptable
carrier in a composition that is formulated as a liquid solution, a sterile and biocompatible
carrier is used. The pharmaceutically acceptable carrier may be physiological saline,
sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose
solution, maltodextrin solution, glycerol, ethanol, or a mixture of two or more thereof.
In addition, the composition of the present invention may, if necessary, comprise
other conventional additives, including antioxidants, buffers, and bacteriostatic
agents.
[0049] The carrier is not limited particularly, but for oral administration, the composition
of the present invention can comprises binders, lubricants, disintegrants, excipients,
emulsifiers, dispersions, stabilizers, suspending agents, pigments, perfumes, etc.,
for injection administration, the composition of the present invention can comprises
buffers, preservatives, analgesics, emulsifiers, isotonic
agents, stabilizers, etc., and for local administration, the composition of the present invention
can comprises bases, excipients, lubricants, preservatives, etc., can be used.
[0050] The inventive composition can be formulated with a pharmaceutically acceptable carrier
as described above in various manners. For example, for oral administration, the composition
of the present invention can be formulated in the form of tablet, troche, capsule,
elixir, suspension, syrup, wafer, etc., and for injection administration, the composition
can be formulated as a unit dosage ampoule or a multiple dosage form. The composition
can also be formulated as solution, suspension, tablet, pill, capsule, sustained-release
formulation, etc.
[0051] In the meantime, examples of carrier, excipient or diluent suitable for the formulation
of the composition may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol,
erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate,
calcium silicate, cellulose, methyl cellulose,
microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium
stearate and mineral oils. In addition, the composition of the present invention may
additionally contain fillers, anti-aggregating agents, lubricants, wetting agents,
perfumes, and preservatives.
[0052] In addition, the pharmaceutical composition of the present invention may include
any one formulation selected from the group consisting of tablets, pills, powders,
granules, capsules, suspensions, internal solutions, emulsions, syrups, sterilized
aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized agents,
and suppositories according to a conventional method.
[0053] In addition, the conjugate may be used in a mixture with various pharmaceutically
acceptable carriers such as physiological saline or organic solvents. To increase
the stability or absorption property of the conjugate, the conjugate may be used in
combination with carbohydrates such as glucose, sucrose or dextran, antioxidants such
as ascorbic acid or glutathione, chelating agents, low-molecular-weight proteins,
or stabilizers.
[0054] In yet another aspect, the present invention is directed to a method of treating
cancer or autoimmune disease using the antibody-drug conjugate or the composition.
The antibody may be an antibody that binds specifically to a cancer cell surface antigen,
and the drug may be a drug for treating cancer. In addition, the antibody may be an
antibody that binds specifically a surface antigen of a tissue in which autoimmune
disease has occurred, and the drug may be a drug for treating autoimmune disease.
[0055] The antibody and the drug are as described above.
[0056] The method may be a method for treatment of cancer or autoimmune disease, which comprises
administering the pharmaceutical composition to a subject in need of the treatment.
The antibody-drug conjugate and carriers that are used in the method are as described
above.
[0057] The composition may be administered as single or multiple doses in a pharmaceutically
effective amount. In this case, the composition may be administered in the form of
liquid, powder, aerosol, capsule, enteric-coated tablet, or suppository. The composition
of the present invention can be administered intraperitoneally, intravenously, intramuscularly,
subcutaneously, transdermally, orally, topically, intranasally, intrapulmonarily or
intrarectally, but is not limited thereto. However, because the protein antibody is
digested when administered orally, the active ingredient in the composition for oral
administration should be coated or formulated so as to be protected from degradation
in the stomach. In addition, the pharmaceutical composition may be administered by
any device by which the active ingredient may be delivered to target cells. Furthermore,
the pharmaceutical composition of the present invention may be administered individually
or in combination with other therapeutic agents, and may be administered sequentially
or simultaneously with conventional therapeutic agents.
[0058] The composition comprising the antibody-drug conjugate of the present invention is
administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically
effective amount" refers to an amount sufficient to treat or prevent the disease at
a reasonable benefit/risk ratio applicable for medical treatment or prevention, and
an effective dosage level can be determined according to the severity of disease,
the activity of the drug, the patient's age, weight, health conditions, gender, and
sensitivity to the drug, time of administration, route of administration and the discharge
rate, duration of treatment, combination with the composition of the present invention
used, or well-known elements and elements in other medical fields, including drugs
used simultaneously.
[0059] In a further aspect, the present invention is directed to a method for screening
an antibody suitable for use in preparation in the antibody-drug conjugate.
[0060] In the preparation of the antibody-drug conjugate according to the present invention,
an antibody suitable for use in effectively preparing the antibody-drug conjugate
by conjugating a drug to the N-terminus (particularly α-amine) of the antibody can
be screened and selected.
EXAMPLES
[0061] Hereinafter, the present invention will be described in further detail with reference
to examples. It will be obvious to a person having ordinary skill in the art that
these examples are illustrative purposes only and are not to be construed to limit
the scope of the present invention. Thus, the substantial scope of the present invention
will be defined by the appended claims and equivalents thereof.
Example 1: Selection of Model Antibodies
[0062] In the preparation of an antibody-cytotoxin conjugate representative of the antibody-drug
conjugate of the present invention, the anti-HER2 antibody Trastuzumab, the anti-CD56
antibody Lorvotuzumab, the anti-CD30 antibody Brentuximab and the anti-GPNMB (glycoprotein
NMB) antibody Glembatumumab were used as model antibodies in order to examine whether
cytotoxin having a linker would be site-selectively conjugated to the antibodies.
[0063] The above antibodies were constructed into expression vectors using known amino acid
sequence information, and a stable cell line was constructed from the CHO cell line.
Alternatively, the antibodies were expressed transiently, incubated and purified.
Example 2: Synthesis of Toxin
[0064] Monomethyl auristatin F (MMAF) toxin having an aldehyde linker connected to the end
was synthesized (LegoChem Biosciences or XcessBioscience) (FIG. 1). In addition, in
order to examine whether the N-terminal conjugation method of the present invention
can also be applied to toxins other than MMAF, Monomethyl auristatin E (MMAE) was
synthesized (XcessBioscience, USA).
Example 3: Preparation of Monoclonal Antibody-Cytotoxin Conjugate
3-1: Preparation of Monoclonal Antibody-Drug Conjugate according to the Present Invention
[0065] An antibody was diluted in 100 mM potassium phosphate buffer (pH 5.49) and concentrated
to about 7.1 mg/ml. Next, MMAF (LegoChem Biosciences, Korea) connected with a linker
having an aldehyde reactive group was dissolved in 50% DMSO (dimethyl sulfoxide) solvent
to a concentration of 2.5 mg/ml. Thereafter, the prepared antibody solution and MMAF
solution were mixed with each other so as to achieve the following conditions: final
70 mM potassium phosphate (pH 6.0); antibody concentration: 5.0 mg/ml; 14% DMSO; MMAF
concentration: 0.3 mg/ml; and the molar ratio between the α-amine of the antibody
and MMAF: about 1:2.3 (or the molar ratio between the antibody and MMAF is 1:9). NaCNBH
3 (Sigma, USA) was added to the reaction solution to a final concentration of 20 mM,
and then reacted at 4°C for 12 hours with gentle stirring. To separate unreacted antibody
and unreacted MMAF connected with the linker, a Sephadex G-25 column (GE Healthcare,
USA) or a resource phenyl column (Resource Phe, GE Healthcare, USA) was used. According
to this process, a conjugate was prepared in which about three MMAF toxin molecules
per antibody molecule were selectively conjugated to the amino terminus of the antibody
(FIG. 2).
3-2: Preparation of Control Antibody-Drug Conjugate
[0066] According to a conventional technology, a control antibody-drug conjugate was prepared
by cysteine conjugation (Thiomab (HC-A114C) + Mal-C6-MMAF), thiol conjugation (Mal-C6-MMAF)
or lysine conjugation (SMCC linker, SH-C6-MMAF).
[0067] To prepare a thiol-conjugated antibody, an antibody was reduced with TCEP at a pH
of 8.0, and then Mal-C4-MMAF was added thereto and allowed to react at 0°C for 3 hours.
After the reaction, thiol was added to the reaction product which was then further
reacted. After termination of the reaction, replacement with 1X PBS using a G25 desalting
column (GE healthcare, USA) was performed to complete the reaction.
[0068] To prepare a cysteine-conjugated antibody, cysteine in a purified antibody was activated,
and then Mal-C6-MMAF was added thereto, and conjugation was performed according to
a process similar to that used in the preparation of the thiol-conjugated antibody.
[0069] A conjugate comprising a lysine-conjugated antibody was prepared with reference to
International Patent Publication No.
WO2005037992 (Immunogen). First, an antibody was reacted with an SMCC linker, and unreacted SMCC
was removed by buffer exchange. The antibody-SMCC conjugate was reacted with SH-C4-MMAF
(Concortis bioscience, USA) containing a thiol group, thereby preparing an antibody-SMCC-MMAF
conjugate.
[0070] The antibody-cytotoxin conjugates prepared by α-amine conjugation according to the
present invention are summarized in Table 1 below.
Table 1
Antibody-drug conjugates |
Conjugation ype |
Conjugation conditions |
Conjugate name (when Trastuzumab and MMAF are used) |
Cys conjugation |
Thiomab (HC A114C), Mal-C6-MMAF |
Thiomab-MMAF |
Thiol conjugation |
Mal-C6-MMAF |
T-C-MMAF |
Lys conjugation |
SMCC linker, SH-C6-MMAF |
T-K-MMAF |
Amine conjugation |
ALD-C6-MMAF (weakly acidic pH) |
T-N-MMAF |
[0071] The four conjugates prepared as described above were analyzed to determine the DAR
(drug antibody ratio) and the site of conjugation. The analysis was performed by LC-MS
and peptide mapping.
Example 4: Physicochemical Properties and Biological Properties
4-1: Analysis of Molecular Weight
[0072] The molecular weights of the antibody-drug conjugates (T-N-MMAF) were determined
by LC-MS analysis. The theoretical molecular weight of the drug (MMAF) used is 824.54
Da, and the molecular weight of Trastuzumab is 145 kDa. Thus, the conjugation of the
drug to the antibody and the number of drug moieties conjugated to one antibody molecule
could be simultaneously determined by mass spectrometry.
[0073] To determine the DAR of T-N-MMAF prepared in Example 3, the molecular weight of T-N-MMAF
was analyzed by LC/MS. The prepared sample was treated with PNGaseF to remove sugar
chains, and then separated through an ACQUITY UPLC BEH 200 SEC column, after which
the sample was injected into the Waters Synapt G2-S system to analyze the mass. The
results of the analysis are shown in FIG. 3.
[0074] As a result, as shown in FIG. 3, chemical species ranging from a chemical species
(D0) having no drug moiety conjugated thereto to a chemical species (D7) having 7
drug moieties conjugated thereto were detected, and the number of drug moieties conjugated
was determined based on whether the difference in molecular weight between peaks was
consistent with or similar to the molecular weight of the drug. The relative intensities
of the drug moieties are shown in Table 2 below. The DAR was calculated as the weighted
average of the chemical species and was DAR=3.2.
Table 2
No. of bound drug |
Mass (Da) |
Relative intensity(%) |
Da |
0 |
145179.5 |
1.8 |
- |
1 |
146005.3 |
10.7 |
825.8 |
2 |
146833.2 |
21.2 |
827.9 |
3 |
147661.1 |
25.4 |
827.9 |
4 |
148488.9 |
21.0 |
827.8 |
5 |
149317.0 |
12.5 |
828.1 |
6 |
150145.5 |
5.0 |
828.5 |
7 |
150964.2 |
2.3 |
818.7 |
DAR |
|
3.219 |
|
DAR = Sum(Intensity(%) x No. of Drug / 100) Drug moiety mass : 828 Da |
|
4-2: Site of Conjugation of Drug
[0075] The site of conjugation of the drug in the prepared T-N_MMAF conjugates was determined
by peptide mapping. T-N-MMAF ADC (having a DAR of 3.2) prepared in Example 3 was treated
with Rapigest (Waters), and then treated with trypsin (Roche) to make fragments. The
reaction product was separated through an ACQUITY UPLC PST (BEH) C18 column, and the
separated peaks were subjected to mass spectrometry through the Waters Synapt G2-S
Q/TOF system to determine the sequence of the reaction product. The results of the
analysis are shown in FIG. 4.
[0076] As a result, as shown in FIG. 4, peaks which were not found in the non-conjugated
parent antibody were detected in the chromatogram. The results of mass spectrometry
indicated that the fragments were the N-terminus of the heavy chain, the N-terminus
of the light chain, a portion of the heavy chain, and other small fragments. The ratios
of the fragments are shown in Table 3 below. Thus, it can be seen that 75% of the
drug was conjugated to the N-terminus and 92% of the drug was selectively conjugated
to the N-terminus and the heavy-chain CH2 region, which can be clearly defined.
Table 3
|
Trypsin fragment |
Ratio |
Heavy chain-N-terminus |
EVQLVESGGGLVQPGGSLR (SEQ ID NO: 1) |
46% |
Light chain-N-terminus |
DIQMTQSPSSLSASVGDR (SEQ ID NO: 2) |
29% |
Heavy chain-CH2 |
THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 3) |
17% |
Others |
- |
8% |
4-3: Analysis of Purity
[0077] To determine the aggregate content of the prepared T-N-MMAF conjugate, purity analysis
of the conjugate was performed by SE-HPLC and SDS-PAGE analysis. Size exclusion chromatography
was used in a TSK-Gel3000SWXL column using PBS as a mobile phase, and SDS-PAGE was
performed using 4-12% Novex NuPAGE gel. The results of the chromatography are shown
in FIG. 5.
[0078] As a result, as shown in FIG. 5, the purity of the monomer was 98.8%, which is suitable
for an efficacy test, and fragmentation or cross-linking was not detected.
4-4: Antigen Binding Activity
[0079] In order to examine the antigen binding activity of the antibody is maintained even
after the drug was conjugated thereto, the antigen binding activity of the drug-conjugated
antibody was measured by a method of measuring surface plasmon resonance using Biacore™.
As a control antibody, a natural antibody was used. The antigen (ErbB2) binding activity
was analyzed using Biacore T200, and each antibody was immobilized on a CM5 sensor
chip (GE healthcare, USA) using an amine coupling kit, after which kD (M) was calculated
by measuring and analyzing on/off rate while injecting ErbB2 at concentrations of
50, 16.67, 5.56, 1.85, 0.62 and 0.21 nM and at a rate of 30 uL/min.
Table 4
Samples |
Antigen binding activity |
Test 1 (10-10 M) |
Test 2 (10-10 M) |
Average (10-10 M) |
Trastuzumab |
1.3 |
2.2 |
1.8 |
T-N-MMAF (DAR 1.6) |
1.2 |
0.9 |
1.1 |
T-N-MMAF (DAR 3.2) |
1.1 |
0.7 |
0.9 |
[0080] As a result, as summarized in Table 4 above, it was found that an antigen binding
activity of about 0.1 nM similar to that of the natural antibody was maintained even
after drug conjugation regardless of the DAR.
Example 5: In vitro Cytotoxicity Analysis
[0081] In order to examine the
in vitro efficacy of the prepared antibody-cytotoxin conjugate, an anti-proliferation assay
was performed using BT474, HCC1954, SKOV-3, JIMT-1 cell lines which are HER2-expressing
tumor cell lines. Each of the cell lines was cultured and suspended at a concentration
of 1 x 10
5 cells/ml, and 100
µℓ of the suspension was loaded into each well of a 96-well plate. The cells were incubated
in an incubator for 3 hours, and then 100
µℓ of the antibody-cytotoxin conjugate diluted to various concentrations was added to
each well of the plate and incubated in an incubator for 4 days. A 1:10 dilution of
CCK-8 (Dojindo) was added to each well of the plate, which was then covered with a
foil and incubated in an incubator for 2-5 hours. Next, the absorbance of each well
at 450 nm was measured using a SpectraMax 190 microplate reader (Molecular Device).
Table 5
Cell line |
Cytotoxicity (IC50 (pM)) |
T-N-MMAF (DAR 3.2) |
T-C-MMAF (DAR 3. 6) |
T-K-MMAF (DAR 3. 9) |
HCC1954 |
40 |
22 |
45 |
SKOV-3 |
104 |
59 |
N.D. |
JIMT1 |
253 |
98 |
727 |
BT474 |
116 |
49 |
77 |
[0082] As a result, as shown in Table 5 below, T-N-MMAF showed cytotoxicity slightly lower
than T-C-MMAF in all the four cancer cell lines, but a significant decrease in cytotoxicity,
which can influence
in vivo efficacy, was not observed.
Example 6: Stability Test
6-1: Stability in Human Serum In vitro
[0083] Using T-N-MMAF prepared in Example 3 and control antibodies, including a natural
antibody, T-C-MMAF and Thiomab-MMAF, a stability test in human serum
in vitro was performed. The antibody-cytotoxin conjugate was buffer-exchanged with 1 x PBS
and concentrated to 3.33 mg/ml, and then mixed with human serum (Sigma, USA)at a ratio
of 1:9 (v/v) and allowed to stand at 37°C for 7 days. After 7 days, to remove proteins
other the stored sample was treated with MabSelectSure (GE healthcare, USA) than the
antibody-cytotoxin conjugate contained in the sample in order to minimize interference
in LC/MS analysis. The stability of the conjugate in human serum
in vitro was analyzed by LC/MS, and the results of the analysis are shown in Table 6 below.
Table 6
Samples |
Relative content of monoclonal antibody (7 days of storage, %) |
Relative content of conjugate (7 days of storage, %) |
Relative content of DAR (7 days of storage, %) |
Trastuzumab |
90.0 |
- |
- |
T-N-MMAF |
89.3 |
90.5 |
101.4 |
T-C-MMAF |
49.2 |
32.3 |
65.6 |
Thiomab-MMAF |
69.9 |
59.5 |
85.1 |
[0084] As a result, as can be seen in Table 6 above, the changes in the content and DAR
of T-N-MMAF compared to the control natural antibody after 7 days of storage were
not observed. However, in the case of T-C-MMAF and Thiomab-MMAF, which are the comparative
antibody-drug conjugates, decreases in the total antibody content and the DAR could
be observed.
6-2: Rat pharmacokinetics (PK)
[0085] In vivo stability was compared and analyzed through a rat pharmacokinetic experiment. Each
of three ADCs (T-K-MMAF, T-C-MMAF, and T-N-MMAF) and Trastuzumab were injected intravenously
once to female Sprague-Dawley rats at a dose of 2.5 mg/kg. At 0.05, 0.5, 1, 6, 24,
72, 168, 240 and 336 hours after administration of the substances, blood was collected.
A total antibody assay of analyzing all antibodies binding to ErbB2 in blood and a
conjugated antibody assay of analyzing an antibody maintaining drug conjugation were
performed by an ELISA method.
[0086] The total antibody content was analyzed by an ELISA method as follows.
[0087] A 96-well microplate was coated with ErbB2 (R&D systems), and then a sample was added
to the plate and incubated at a temperature of 37°C for 1 hour. The plate was washed
with PBST to remove all non-fixed substances, and then the absorbance of the plate
at 450 nm was measured using HRP-conjugated anti-human kappa light chain antibody
and 3,3',5,5'-tetramethylbenzidine (TMB, Sigma, T0440), thereby determining the total
antibody content of the sample.
[0088] The conjugated antibody assay was performed by a method similar to the above-described
method. Specifically, a 96-well microplate was coated with anti-MMAF antibody (Young
In Frontier), and then a sample was added to the plate and incubated at a temperature
of 37°C for 1 hour. Next, biotinylated ErbB2 (ACROBIOSYSTEMS, USA), streptavidin-HRP
and TMB were sequentially added to the plate to develop color, and then the absorbance
of the plate at 450 nm was measured to determine the concentration of the conjugated
antibody. The results of the measurement are shown in FIGS. 6 to 8 and Table 7 below.
Table 7
PK parameters measured after administering ADCs to rats at a dose of 2.5 mg/kg |
Treatment (n=5, each) 2-compartment modeling |
Total Ab |
Conjugated Ab |
AUC (hr*µg/ml ) |
T1/2 (hr) |
Cmax (µg/ml) |
AUC (hr*µg/ml ) |
T1/2 (hr) |
Cmax (µg/ml ) |
Trastuzumab |
6964.9 |
115.7 |
128.1 |
- |
- |
- |
T-N-MMAF |
6795.7 |
122.1 |
111.2 |
6813.2 |
118.3 |
112.4 |
T-C-MMAF |
5933.5 |
111.6 |
94.3 |
4315.4 |
84.6 |
93.7 |
T-K-MMAF |
4324.3 |
65.1 |
157.5 |
3781.7 |
53.2 |
190.0 |
Example 7: Test for Anticancer Effect in Anticancer Model Animals
[0089] In order to examine the efficacy of three ADCs prepared by different techniques and
the difference in efficacy by drug-antibody ratio (DAR), an
in vivo efficacy test was performed in breast cancer (HCC 1954) xenograft models using nude
rats.
[0090] Each of four ADCs, that is, T-N-M (DAR: about 1.6 and 3.2), T-C-M (DAR: about 3.7)
and T-K-M (DAR: about 3.9), was administered intravenously once to HCC1954 cell-transplanted
rats at a dose of 1 mg/kg, and then the degree of inhibition of growth of the transplanted
tumor was compared between the test groups. The results are shown in FIGS. 9 and 10.
[0091] As a result, as shown in FIGS. 9 and 10, the antibody according to the present invention
had an excellent anticancer effect compared to the control and comparative antibodies.
Example 8: Toxicity Test
[0092] In order to examine whether stability varying depending on the technique for preparation
of ADCs influences toxicity, a single-dose toxicity test was performed using SD rats.
Each of three ADCs was administered intravenously once at a high dose of 200 mpk.
As comparative groups, an antibody alone and MMAF were administered at a dose of 200
mpk. The weight was measured everyday during a period ranging from the time point
of administration of the test substance to the end of the test (day 12). Biochemical
analysis of the blood was performed at 5 days after administration. Measurement items
were AST and ALT for determining hepatotoxicity and typical hematological toxicity,
neutrophils and platelets.
8-1: Change in Weight
[0093] The results of measurement of changes in the weight are shown in FIG. 11. As shown
in FIG. 11, the T-C-MMAF and T-K-MMAF groups showed a distinct decrease in the weight
compared to the T-N-MMAF group and other groups. In particular, in the case of the
group administered with T-C-MMAF, all animals excluding one animal did die after day
8.
8-2: Biochemical Analysis (Hepatotoxicity)
[0094] In order to examine whether the ADCs cause hepatotoxicity, biochemical analysis of
blood collected at day 5 after administration of the ADCs was performed. The analysis
was performed using the Au480 clinical analyzer (Beckman Coulter, USA), and the levels
of AST (aspartate aminotransferase) and ALT (alanine aminotransferase) indicative
of hepatotoxicity were measured. The results of the measurement are shown in FIG.
12.
[0095] As a result, as shown in FIG. 12, it could be observed that the T-N-MMAF group according
to the present invention showed no significant difference from other control groups
including PBS, indicating that it did not caused abrupt or serious hepatotoxicity.
However, a significant increase in AST and ALT was observed in the T-C-MMAF and T-K-MMAF
groups, indicating that administration of the drugs caused hepatotoxicity.
8-3: Hematological Analysis (Neutropenia and Thrombocytopenia)
[0096] Because the major clinical toxicities of currently approved ADCs indicate the hematological
properties, hematological analysis of blood collected at day 5 after administration
of the ADCs was performed using the Hemavet 950 FS hematological analyzer (Drew Scientific
Inc., USA). The results of the analyzer are shown in FIG. 13.
[0097] As a result, as shown in FIG. 13, the T-N-MMAF group showed no significant change
in the number of neutrophils compared to the control groups including PBS, suggesting
that T-N-MMAF did not cause abrupt and serious hematological toxicity. However, the
T-C-MMAF group showed a significant decrease in the number of neutrophils, and the
T-K-MMAF group showed a significant increase in the number of neutrophils, which decreased
immediately after administration and then increased. Thus, for these two groups, it
could be concluded that abrupt hematological toxicity was caused by administration
of the drugs.
[0098] The number of platelets was noticeably smaller than in the T-N-MMAF group than in
other control groups including PBS. However, the T-C-MMAF and T-K-MMAF groups showed
a significant decrease in the number of platelets, indicating that abrupt toxicity
was caused by administration of the drugs.
Example 9: Examination of Platform Function
[0099] Whether the method for preparing the antibody-drug conjugate according to the present
invention can be applied to various antibody-drug conjugates was examined. For this,
the method was applied to various drugs or antibodies and various antibody forms in
order to examine the function thereof.
9-1: Examination of Function according to Type of Drug
[0100] In order to determine whether the method for preparing the antibody-drug conjugate
according to the present invention can be applied to various drugs, N-terminal conjugation
of various drugs was performed using Trastuzumab as a model antibody. Specifically,
two drugs (MMAF and MMAE) were used, and the results obtained using MMAF are as described
in the Examples above. Antibody-drug conjugates were prepared according to the method
described in Example 1, and the DAR analysis,
in vitro stability and rat PK of the prepared antibody-drug conjugates were performed according
to the methods described in the Examples above.
9-1-1: Preparation of T-N-MMAE
[0101] According to a conjugate between MMAE (XcessBioscience, USA) and an antibody was
prepared. To determine the DAR of the conjugate, the molecular weight of the conjugate
was analyzed by LC/MS, and the results of the analysis are shown in FIG. 14 and Table
8 below.
Table 8
Chemical species distribution by DAR of T-N-MMAE and average DAR |
No. of drug |
Mass (Da) |
Relative content (%) |
Delta mass |
D0 |
N/D |
N/D |
|
D1 |
146061.05 |
6.3 |
|
D2 |
146863.77 |
14.3 |
802.72 |
D3 |
147672.80 |
23.3 |
809.03 |
D4 |
148484.33 |
24.2 |
811.53 |
D5 |
149297.30 |
16.5 |
812.97 |
D6 |
150112.25 |
8.6 |
814.95 |
D7 |
150922.84 |
6.8 |
810.59 |
DAR |
|
3.83 |
|
9-1-2: Analysis of Stability of T-N-MMAE in Human Serum
[0102] According to the method of Example 6, the stability of T-N-MMAE ADC in serum was
evaluated. The concentration of ADC in each sample was measured by the total antibody
assay using ELISA, and a change in the DAR was measured by LC/MS.
Table 9
|
µg/ml |
% |
DAR |
% |
Day 0 |
364.8 |
100% |
3.17 |
100% |
Day 3 |
346.9 |
95% |
3.33 |
105% |
Day 7 |
294.8 |
81% |
3.28 |
103% |
9-1-3: Rat PK of T-N-MMAE
[0103] In order to evaluate the
in vivo stability of the prepared MMAE conjugate, a PK study in SD rats was performed according
to a method similar to that of Example 6. Shortly, 2.5 mg/pk of the ADC was administered
to female SD rats. At 12 min, 30 min, 1 hour, 6 hour, 24 hours, 3 days, 7 days, 10
days, 14 days, 17 days and 21 days after administration of the ADC, blood was collected
from the rats, and the concentrations of total protein and conjugated antibody in
the blood were measured according to the above-described methods using an ELISA technique.
Table 10
Rat PK parameters of T-N-MMAE |
Group |
AUC (hr*µg/ml) |
Conjugate /Total ratio |
half-life (hr) |
Conjugate /Total ratio |
trastuzumab |
5868.83 |
|
169.6 |
|
T-N-MMAF (T) |
6688.95 |
|
191.8 |
|
T-N-MMAF (C) |
6871.71 |
103% |
203.3 |
106% |
T-N-MMAE (T) |
5639.24 |
|
173.9 |
|
T-N-MMAE (C) |
5690.96 |
101% |
163.7 |
94% |
* Trastuzumab and T-N-MMAF were included for comparison between tests. |
[0104] As a result, as shown in FIG. 15 and Table 10 above, T-N-MMAE showed the profiles
of total antibody and conjugated antibody, which did not significantly differ from
that of the parent antibody, suggesting that the antibody-drug conjugate prepared
using MMAE has stability similar to that of the antibody -drug conjugate prepared
using MMAF.
9-1-4: Activity of T-N-MMAE
[0105] In order to determine the biological activity of the prepared MMAE conjugate, the
activity thereof was measured using four different tumor cell lines. The results of
the measurement are shown in Table 11 below. The method used was similar to that used
in Example 5.
Table 11
Cytotoxicity of T-N-MMAE for HER2-expressing tumor cell lines |
|
IC50 [nM] |
#1 |
#2 |
Average |
HCC1954 |
0.39 |
0.26 |
0.33 |
SKOV-3 |
3.04 |
2.62 |
2.83 |
JIMT1 |
4.00 |
3.51 |
3.76 |
BT474 |
0.56 |
0.70 |
0. 63 |
[0106] As a result, the measured IC
50 was in the range of 0.33-3.76 nM, which was similar to the activity (0.47 nM) of
the BT474 cell line against the Trastuzumab/MMAE thiol conjugate reported in the literature.
This suggests that the method for selective conjugation to the N-terminal α-amine
according to the present invention can also be applied to other types of drugs.
9-2: Examination of Function according to Type of Antibody
[0107] In order to examine whether the method for preparing the antibody-drug conjugate
according to the present invention can be applied to various antibodies, N-terminal
conjugation to three anticancer antibodies (Brentuximab, Lorvotuzumab, Glembatumumab)
was performed, and the DAR and
in vitro stability of the conjugates were measured.
9-2-1: Brentuximab
9-2-1-1: Preparation of Brentuximab-N-MMAF
[0108] Using Brentuximab expressed from the CHO cell line, Brentuximab-N-MMAF (B-N-MMAF)
was prepared according to the method of Example 3. The prepared ADC showed the LC/MS
profile shown in FIG. 16 and Table 12 below. In the ADC, chemical species ranging
from D0 to D6 were detected, and the DAR was calculated to be 2.90.
Table 12
No. of bound drug |
Mass (Da) |
Relative content (%) |
Delta mass (Da) |
D0 |
145208.6 |
3.1 |
|
D1 |
146034.9 |
14 |
826.3 |
D2 |
146863.3 |
24.4 |
828.4 |
D3 |
147692 |
26.4 |
828.7 |
D4 |
148520.7 |
18.2 |
828.7 |
D5 |
149349.7 |
9.3 |
829 |
D6 |
150177.5 |
4.7 |
827.8 |
DAR |
|
2.90 |
|
9-2-1-2: Ligand Binding Assay
[0109] In order to determine whether the properties of the antibody are changed by conjugation,
the activity of binding of the antibody to an antigen was measured by an ELISA technique.
Specifically, 100 µg of the antigen CD30 (R&D Systems) was coated on a 96-well microplate,
and then blocked with 1% BSA at 37°C for 1 hour. After the blocking solution was removed,
a sample was added to the plate and incubated at 37°C for 1 hour. The plate was washed
five times with PBST (PBS + 0.05% tween 20), and then a 1000-fold dilution of HRP-conjugated
anti-human kappa light-chain antibody was added to the plate and incubated at 37°C
for 1 hour. The plate was washed five times with PBST, and then TMB (Sigma) was added
to the plate which was then subjected to color development for 10 minutes. IN H
2SO
4 was added to the plate to stop the reaction, and then the absorbance of the plate
at 450 nm was measured. The results of the measurement are shown in FIG. 17. In FIG.
17, the line indicated by 0 indicate results for non-conjugated Brentuximab, the line
indicated by ◇ indicates results for B-N-MMAF having a DAR of 2.90, and the line indicated
by Δ indicates results for B-N-MMAF having a DAR of 4.22. As can be seen from the
results, the activity of binding of the antibody to the antigen did not change even
after conjugation regardless of the DAR value.
9-2-1-3: In vitro Cytotoxicity
[0110] To determine the
in vitro efficacy of the prepared antibody-cytotoxin conjugate, an anti-proliferation assay
was performed using Karpas-299 and L-540 cell lines that are CD30-expressing cell
lines.
[0111] Specifically, each of the cell lines was cultured and suspended at a concentration
of 1 x 10
5 cells/ml, and 100
µℓ of the suspension was loaded into each well of a 96-well plate. The cells were incubated
in an incubator for 3 hours, and then 100
µℓ of the antibody-cytotoxin conjugate diluted to various concentrations was added to
each well of the plate which was then incubated in an incubator for 4 days. A 1:10
dilution of CCK-8 (Dojindo) was added to each well of the plate, which was then covered
with a foil and incubated in an incubator for 2-5 hours. Next, the absorbance of each
well at 450 nm was measured using a SpectraMax 190 microplate reader. The results
of the measurement are shown in Table 13 below.
Table 13
Cell line |
IC50(pM) |
Karpas-299 |
32.2 |
L-540 |
37.1 |
[0112] As a result, a cytotoxicity lower than 40 pM was observed in all the two cell lines
(Karpas-299 and L-540).
9-2-2: Lorvotuzumab
9-2-2-1: Preparation of Lorvotuzumab-N-MMAF
[0113] Using Lorvotuzumab expressed transiently from CHO cells, Lorvotuzumab-N-MMAF (L-N-MMAF)
was prepared according to the method of Example 3. As a result, the prepared ADC showed
the conjugation profile shown in FIG. 18 and Table 14 below, and the DAR of the conjugate
was determined to be 3.33.
Table 14
No. of bound drugs |
Mass (Da) |
Relative content (%) |
Delta mass (Da) |
D0 |
147001.5 |
3.3 |
|
D1 |
147830.8 |
10.8 |
829.3 |
D2 |
148657.9 |
18.6 |
827.1 |
D3 |
149486.6 |
22.7 |
828.7 |
D4 |
150315.4 |
20.1 |
828.8 |
D5 |
151144.7 |
13.7 |
829.3 |
D6 |
151973.5 |
7 |
828.8 |
D7 |
152803 |
3.7 |
829.5 |
DAR |
|
3.329 |
|
9-2-2-2: Ligand Binding Assay
[0114] In order to determine whether the properties of the antibody are changed by conjugation,
the activity of binding of the antibody to an antigen before and after conjugation
was measured by an ELISA technique. Specifically, 100 µg of the antigen CD30 (R&D
Systems, 2408-NC-050) was coated on a 96-well microplate at a concentration of 1 µg/ml,
and then blocked with 1% BSA at 37°C for 1 hour. After the blocking solution was removed,
a test sample was added to the plate and incubated at 37°C for 1 hour. The plate was
washed five times with PBST (PBS + 0.05% tween 20), and then a 1000-fold dilution
of HRP-conjugated anti-human kappa light-chain antibody was added to the plate and
incubated at 37°C for 1 hour. The plate was washed five times with PBST, and then
TMB (Sigma) was added to the plate which was then subjected to color development for
10 minutes. IN H
2SO
4 was added to the plate to stop the reaction, and then the absorbance of the plate
at 450 nm was measured. The results of the measurement are shown in FIG. 19. In FIG.
19, the line indicated by O indicate results for the antibody not conjugated to the
drug, the line indicated by Δ indicates results for L-N-MMAF having a DAR of 2.5,
and the line indicated by ◇ indicates results for L-N-MMAF having a DAR of 3.3. As
can be seen from the results, the activity of binding of the antibody to the antigen
was maintained regardless of the DAR value.
9-2-2-2: In vitro Cytotoxicity
[0115] To determine the
in vitro efficacy of the prepared antibody-cytotoxin conjugate, an anti-proliferation assay
was performed using an OPM-2 cell line. Specifically, the cell line was cultured and
suspended at a concentration of 1 x 10
5 cells/ml, and 100
µℓ of the suspension was loaded into each well of a 96-well plate. The cells were incubated
in an incubator for 3 hours, and then 100
µℓ of the antibody-cytotoxin conjugate diluted to various concentrations was added
to each well of the plate which was then incubated in an incubator for 4 days. A 1:10
dilution of CCK-8 (Dojindo) was added to each well of the plate, which was then covered
with a foil and incubated in an incubator for 2-5 hours. Next, the absorbance of each
well at 450 nm was measured using a SpectraMax 190 microplate reader. The results
of the measurement are shown in Table 15 below.
Table 15
IC50 [nM] |
OPM-2 |
L-N-MMAF DAR 2.5 |
52.9 |
L-N-MMAF DAR 3.3 |
41.9 |
[0116] As can be seen in Table 15 above, the L-N-MMAF antibody according to the present
invention showed a cytotoxicity of about 42-53 nM.
9-2-3: Glembatumumab
9-2-3-1: In vitro Cytotoxicity
[0117] To determine the
in vitro efficacy of the prepared antibody-cytotoxin conjugate, an anti-proliferation assay
was performed using a SK-MEL-2 cell line that is a skin cancer cell line. Specifically,
the cell line was cultured and suspended at a concentration of 1 x 10
5 cells/ml, and 100
µℓ of the suspension was loaded into each well of a 96-well plate. The cells were incubated
in an incubator for 3 hours, and then 100
µℓ of the antibody-cytotoxin conjugate diluted to various concentrations was added
to each well of the plate which was then incubated in an incubator for 4 days. A 1:10
dilution of CCK-8 (Dojindo) was added to each well of the plate, which was then covered
with a foil and incubated in an incubator for 2-5 hours. Next, the absorbance of each
well at 450 nm was measured using a SpectraMax 190 microplate reader. The results
of the measurement are shown in Table 16 below.
Table 16
SK-MEL-2 |
IC50 (nM) |
G-N-MMAF DAR 2.2 |
5.47 |
G-N-MMAF DAR 3.4 |
3.36 |
[0118] As can be seen in Table 16 above, the G-N-MMAF according to the present invention
showed a cytotoxicity of about 3-5 nM.
[0119] The above-described results suggest that a new platform of the antibody-drug conjugate
prepared by site-specific conjugation of the drug to the N-terminal amino acid residue
of the heavy chain or light chain of the antibody shows no reduction in the target
specificity of the antibody while having high stability and also that the therapeutic
effect of the antibody can be doubled by the drug conjugated thereto.
1. An antibody-drug conjugate comprising a cytotoxic drug conjugated to the N-terminal
amino acid residue of the heavy chain or light chain of an antibody.
2. The antibody-drug conjugate of claim 1, wherein the cytotoxic drug is conjugated to
the N-terminal α-amine group of the heavy chain or light chain of the antibody.
3. The antibody-drug conjugate of claim 2, wherein the cytotoxic drug is conjugated to
the antibody by a reactive group of the cytotoxic drug, which is capable of crosslinking
with the α-amine group.
4. The antibody-drug conjugate of claim 3, wherein the reactive group capable of crosslinking
with the α-amine group is selected from the group consisting of isothiocyanate, isocyanate,
acyl azide, NHS ester, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate,
aryl halide, imidoester, carbodiimide, anhydride, and fluorophenyl ester.
5. The antibody-drug conjugate of claim 1, wherein the antibody includes full-length
antibodies or antibody fragments containing antigen binding domains.
6. The antibody-drug conjugate of claim 5, wherein the antibody is selected from the
group consisting of IgG, scFv, Fv, Fab, Fab', and F(ab')2.
7. The antibody-drug conjugate of claim 1, wherein the cytotoxic drug is selected from
the group consisting of microtubule structure formation inhibitors, meiosis inhibitors,
RNA polymerase inhibitors, topoisomerase inhibitors, DNA intercalators, DNA alkylators,
ribosome inhibitors, radioisotopes, and toxins.
8. The antibody-drug conjugate of claim 7, wherein the cytotoxic drug is selected from
the group consisting of maytansinoid, auristatin, dolastatin, tubulysin, calicheamicin,
pyrrolobenzodiazepines, doxorubicin, duocamycin, carboplatin(paraplatin), cisplatin,
cyclophosphamide, ifosfamide, nidran, [nitrogen mustard(mechlorethamine HCL)], bleomycin,
mitomycin C, cytarabine, fluorouracil, gemcitabine, trimetrexate, methotrexate, etoposide,
vinblastine, vinorelbine, alimta, altretamine, procarbazine, taxol, taxotere, topotecan,
irinotecan, trichothecene, CC1065, alpha-amanitin, other enediyne antibiotics, exotoxin,
and plant toxin.
9. The antibody-drug conjugate of claim 8, wherein the auristatin is monomethyl auristatin
E or monomethyl auristatin F.
10. The antibody-drug conjugate of claim 1, wherein the antibody binds specifically to
a cancer cell surface antigen.
11. The antibody-drug conjugate of claim 10, wherein the cancer cell surface antigen is
selected from the group consisting of CD19, CD20, CD30, CD33, CD37, CD22, CD56, CD70,
CD74, CD138, Muc-16, mesothelin, HER2, HER3, GPNMB(glycoprotein NMB), IGF-1R, BCMA(B
cell maturation antigen), PSMA(prostate-specific membrane antigen), EpCAM(Epithelial
cell adhesion molecule), and EGFR (epidermal growth factor receptor).
12. The antibody-drug conjugate of claim 1, wherein the antibody is selected from the
group consisting of an anti-HER2 antibody, an anti-CD30 antibody, an anti-CD56 antibody,
and an anti-GPNMB (glycoprotein NMB) antibody.
13. The antibody-drug conjugate of claim 12, wherein the antibody is selected from the
group consisting of Trastuzumab, Lorvotuzumab, Brentuximab, and Glembatumumab.
14. An antibody-drug conjugate comprising an immunosuppressive agent conjugated to the
N-terminal amino acid residue of the heavy chain or light chain of an antibody.
15. A method for preparing the antibody-drug conjugate of any one of claims 1 to 14, the
method comprising conjugating the cytotoxic drug or the immunosuppressive agent to
the N-terminal α-amine group of the heavy chain or light chain of the antibody, by
allowing an antibody to react with a cytotoxic drug or an immunosuppressive agent
containing a reactive group capable of crosslinking with an α-amine group.
16. The method of claim 15, further comprising separating the antibody-drug conjugate
from a reaction product including the antibody and the cytotoxic drug or the immunosuppressive
agent, in which the conjugate is not formed.
17. The method of claim 15, wherein the reactive group capable of crosslinking with the
α-amine group is selected from the group consisting of isothiocyanate, isocyanate,
acyl azide, NHS ester, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate,
aryl halide, imidoester, carbodiimide, anhydride, and fluorophenyl ester.
18. A pharmaceutical composition for treating cancer, which comprises the antibody-drug
conjugate of any one of claims 1 or 13.
19. A pharmaceutical composition for treating autoimmune disease, which comprises the
antibody-drug conjugate of claim 14.
20. A method for treating cancer, which comprises administering the antibody-drug conjugate
of any one of claims 1 or 13 to a subject suspected of having cancer.
21. A method for treating autoimmune disease, comprising administering the antibody-drug
conjugate of claim 14 to a subject suspected of having autoimmune disease.